FAK purification

Introduction
Production and purification of recombinant FAK (Ptk2). his-tagged FAK gene is in a pFastBac vector. We produce recombinant
baculovirus in SF9 cells using the Bac-to-Bac Baculovirus Expression System. Optimal time to harvest cells after P1 infection
should be optimized to minimize FAK degredation (typically 24 - 48 hours). Note that FAK will phase separate and agregate at
low salt, so we do not do Anion Exchange Chromatography step. This requires starting material to be fairly pure, and not have too many degredation products.

Materials
› Pellets expressing his-FAK
› Lysis Buffer
   25 mM Hepes pH 7.5, 20 mM Imidazole pH 7.0, 500 mM NaCl, 10% glycerol, 5mM BME, 1 mM Benzamadine
› Wash Buffer
   25 mM Hepes pH 7.5, 20 mM Imidazole pH 7.0, 500 mM NaCl, 10% glycerol, 5mM BME, 1 mM Benzamadine
› Elution Buffer
   25 mM Hepes pH 7.5, 400 mM Imidazole pH 7.0, 1 M NaCl, 10% glycerol, 5 mM BME, 1 mM Benzamadine
› Gel Fitration Buffer (GFB)
   25 mM Hepes pH 7.5, 300 mM NaCl, 1 mM DTT, 10% glycerol
› Ni-NTA Resin
› SD200 Solumn
› Roche cOmplete EDTA-free Protease Inhibitor Tablet
› Dounce Homogenizer

Procedure
Day 1
1. Thaw pellets. Add 500 mM NaCl, 5 mM BME and 1 protease inhibitor tablet.
2. If pellets are too concentrated, dilute a bit with Wash Buffer. The more concentrated it is, the more viscous and harder it is to pipette after lysis.
3. Lyse cells with Dounce homogenizer. When using a Dounce, be slow and gentle with your movements to avoid bubbles. 3-5 passes with size A pestle followed by 3-5 passes with size B pestle is sufficient.
4. Pellet 20,000 rpm for 45 min. Pellets will be very soft, so be careful when collecting from centrifuge. DO NOT pour off supernatent. Rather, use a pipette to carefully remove supernatent.
5. Meanwhile, rinse NiNTA resin (typically use 5-10 mL resin) with ~150 mL of water and 25 mL of Wash Buffer.
6. Carefully pipette supernatent and add to equilibrated Ni-NTA resin. Incubate for at least 20 min with gentle rocking or rotating at 4°C.
 

7. Collect flow through.
8. Wash with at least 400 mL Wash Buffer.
9. Elute with 100 mL Elution Buffer (2 x 50 mL fractions).
10. Run gel to confirm protein in eluate.
Ideally, FAK Eluate will be clean and ready for Gel Filtration. However, if the prep is less clean, it can still be used to make fluorescently labeled FAK. During the labeling protocol, FAK goes over two gel filtration columns, which is usually sufficient to reach 80-90% purity.
11. Remove his tag with TEV protease with gentle rocking or rotating overnight at 4°C.
12. PAUSE Meanwhile, equilibrate large SD200 column in GFB overnight.

Day 2
13. Concentrate cleaved FAK in 30K MW cutoff concentrator. (Optional: Run gel to confirm cleavage).
14. Filter concentrated protein and run over SD 200 (make sure final volume is appropriate for the column you are using.
15. Run gel to determine which fractions to keep.
16. Pool fractions, concentrate to at least 20 μM (A280 ~3), aliquot into pcr tubes, flash freeze in LN2, and store aliquots in -80°C freezer.